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Abstract

Mechanical ventilation (MV) of patients can cause damage to bronchoalveolar epithelium, leading to a sterile inflammatory response, infection and in severe cases sepsis. Limited knowledge is available on the effects of MV on the innate immune defense system in the human lung. In this study, we demonstrate that cyclic stretch of the human bronchial epithelial cell lines VA10 and BCi NS 1.1 leads to down-regulation of cathelicidin antimicrobial peptide (CAMP) gene expression. We show that treatment of VA10 cells with vitamin D3 and/or 4-phenyl butyric acid counteracted cyclic stretch mediated down-regulation of CAMP mRNA and protein expression (LL-37). Further, we observed an increase in pro-inflammatory responses in the VA10 cell line subjected to cyclic stretch. The mRNA expression of the genes encoding pro-inflammatory cytokines IL-8 and IL-1β was increased after cyclic stretching, where as a decrease in gene expression of chemokines IP-10 and RANTES was observed. Cyclic stretch enhanced oxidative stress in the VA10 cells. The mRNA expression of toll-like receptor (TLR) 3, TLR5 and TLR8 was reduced, while the gene expression of TLR2 was increased in VA10 cells after cyclic stretch. In conclusion, our in vitro results indicate that cyclic stretch may differentially modulate innate immunity by down-regulation of antimicrobial peptide expression and increase in pro-inflammatory responses.

Introduction

Mechanical ventilation (MV) is a lifesaving treatment for patients suffering from severe respiratory failure by alleviating the work of breathing and facilitating alveolar gas exchange (Slutsky & Ranieri, 2013). MV has, however, been associated with side effects including ventilator induced lung injury (VILI) coupled with injury on lung tissue, stress on epithelial and endothelial barriers, apoptosis, pro-inflammatory responses, increased oxidative stress and secondary infections like nosocomial bacterial pneumonia. This can be followed by sepsis or systemic inflammatory response syndrome and increased mortality (Baudouin, 2001; Uhlig, 2002; Syrkina et al., 2008). Success of treatment with MV requires limitation of VILI and associated side effects (Fan, Villar & Slutsky, 2013). This can be accomplished by either decreasing mechanical stress produced by MV or by increasing the endurance of lung tissues to such strain. Hence, it has become imperative to study the molecular mechanisms behind VILI in details to improve outcomes in patients treated with MV. Although poorly defined, down-regulation of innate immune responses has been proposed to favor bacterial growth and development of ventilator associated pneumonia (VAP) in the lungs of patients during MV (Santos et al., 2005).

Materials and Methods

Cell culture, reagents and cyclic stretch

An E6/E7 viral oncogene immortalized human bronchial epithelial cell line VA10 was cultured as described previously (Halldorsson et al., 2007). Briefly, the cells were maintained in Bronchial/Tracheal Epithelial cell growth medium (Cell Applications, San Diego, CA, USA) with Penicillin-Streptomycin ((20 U/ml, 20 µg/ml, respectively) (Life Technologies, Carlsbad, CA, USA)) at 37 °C and 5% CO2. BCi. NS 1.1 (henceforth referred to as BCi) is a human bronchial epithelial cell line was a kind gift from Dr. Matthew S. Walters, Weill Cornell Medical College, New York NY, USA (Walters et al., 2013) and was established by immortalization with retrovirus expressing human telomerase (hTERT). The BCi cells were cultured as described above for VA10 cell line. Equal amount of cells were seeded on each well in a 6 well collagen I coated Bioflex plates (Flexcell International Corporation, Burlington, CA, USA), and grown to approximately 80% confluence. These plates were then transferred to a base plate of the cell stretching equipment Flexcell FX-5000TM Tension System (Flexcell International Corporation, Burlington, CA, USA) in a humidified incubator at 37 °C and 5% CO2. The cells were subjected to cyclic mechanical stretch with the following parameters: a stretching rate of 20% with a square signal, 0.33 Hz frequency (20 cycles/min) and a 1:1 stretch:relaxation ratio, as described previously (Pugin et al., 2008). The cells were stretched for 6 h and 24 h as described in the results. Control Bioflex plates were kept in the same incubator under static conditions as non-stretch controls. Vitamin D3 (1,25D3) and Sodium 4-phenyl butyric acid (PBA) were purchased from Tocris bioscience, UK. Vitamin D3 was reconstituted in 100% ethanol as per manufacturer’s instructions. The final concentration of the solvent was kept at 0.2% v/v and did not affect gene and protein expression of target genes. PBA was reconstituted in ultrapure H2O.

RNA isolation and quantitative real time PCR

Total RNA was isolated with NucleoSpin RNA kit (Macherey-Nagel, Düren, Germany) and quantified on a spectrophotometer (Nanodrop, Thermo Scientific, Waltham, MA, USA). One µg of total RNA was reverse transcribed into first strand cDNA for each sample with a RevertAid First strand cDNA synthesis kit (Thermo Scientific, Waltham, MA, USA) and modified with 100 unit of reverse transcriptase per reaction. Power SYBR® green Universal PCR master mix (Life technologies, Carlsbad, CA, USA) was used to quantify the cDNA on a 7500 Real time PCR machine (Life technologies, Carlsbad, CA, USA). The reference gene used for all experiments was UBC (Ubiquitin C) and PPIA (Peptidylprolyl Isomerase A) and an arithmetic mean of reference gene Ct values was used. Primers for TLR1 and TLR6 were designed with Pearl primer and used at a final concentration of 300 nM (Marshall, 2004). All other primers were purchased from Integrated DNA technologies (PrimeTime Predesigned qPCR Assay) and used at a final concentration of 500 nM as per manufacturer’s instructions. The qPCR cycling conditions were as follows; (1) Hold stage: 95 °C for 10 min, followed by 40 cycles of (2) De-natured stage: 95 °C for 15 s and (3) Annealed/extended stage: 60 °C for 1 min. The 2(−ΔΔCT) Livak method was utilized for calculating fold differences over untreated control (Livak & Schmittgen, 2001). A detailed list of the primers used in the q-RT-PCR assay is shown in Table 1.

Oxidative stress measurement

After subjecting VA10 cells to cyclic stretch, CellROX green reagent (Life Technologies, Carlsbad, CA, USA) was added to the medium at a final concentration of 5 µM for 30 min. Next, the cells were washed twice with cold 1× PBS (1,000 rpm for 5 min) and detached with a 1× Accutase solution (Millipore, Billerca, MA, USA). Cells were then harvested and suspended in 100 µl of MACS buffer. (Miltenyi Biotec, San Diego, CA, USA) as per manufacturer’s instructions. The samples were analyzed in MACSQuant flow cytometer (Miltenyi Biotec, San Diego, CA, USA), placing the CellROX green reagent signal in FL1. Intact cells were gated in the Forward Scatter/Side Scatter plot to exclude debris. The resulting FL1 data was plotted on a histogram and is represented as % CellROX positive cells before and after cyclic stretch.

Statistical analysis

The q-RT PCR and ELISA results are represented as means ± standard errors of the means (S: E.) from three independent experiments. An unpaired Student’s t-test was used to compare two samples. P < 0.05 was considered statistically significant. All the statistical analysis for q-RT PCR and ELISA experiments was performed with the Prism 6 software (Graph Pad, USA). The Western blot and immunofluorescence data are represented from at least three independent experiments showing similar results.

Results

Cyclic stretch down-regulates the expression of the cathelicidin antimicrobial peptide

We screened for the effect of mechanical stretch on AMP expression. VA10 cells were subjected to stretch for 6 and 24 h to analyze early and late changes in AMP mRNA expression. The mRNA expression of AMPs cathelicidin (CAMP), human beta defensin-1 (DEFB1), Lactoferrin (LTF) and Lysozyme (LYZ) was analyzed with quantitative real time PCR (qRT-PCR). The basal mRNA expression of CAMP was decreased at both 6 h and 24 h after cell stretching (Fig. 1A). DEFB1 mRNA expression was reduced at 24 h after cell stretching but was unaffected after 6 h (Fig. S1). The basal mRNA expression of LTF and LZY was very low (Ct > 32) in the VA10 cells and was excluded from this study. The decrease in cathelicidin gene expression was further confirmed at protein level with Western blot (Fig. 1B) and immunofluorescence analysis (Fig. 1C). Western blot analysis of stretched VA10 cells showed a decrease in secreted pro-LL-37 (encoded by the CAMP gene) levels after 24 h of cyclic stretch (Fig. 1B). Further, immunofluorescence staining of stretched VA10 cells also showed a decrease in LL-37 protein expression at both 6 h and 24 h after stretching (Fig. 1C).

(A) VA10 cells were stretched for 6 and 24 h. The mRNA expression of cathelicidin antimicrobial peptide (CAMP) was analyzed with q-RT PCR after cell stretching (n = 3, mean ± S.E.). Relative expression levels (y-axis) in static cells were defined with an arbitrary value of ‘1’ and changes relative to this value in stretched samples are represented. (B) VA10 cells were subjected to cyclic stretch for 24 h. Cultured supernatants from stretched cells were used for analysis of secreted cathelicidin (pro-LL-37) protein expression by Western blot. Total protein loading is shown by staining with MemCode blue protein stain. The Western blot is a representative of three independent experiments showing similar results. (C) VA10 cells were stretched for 6 h and 24 h. The cells were then stained with antibody against LL-37 (green) and protein expression was visualized with immunofluorescence confocal microscopy. The cells were counterstained with nuclear stain DAPI (blue). Data is representative of three independent experiments showing similar results. Bar = 40 µm (ns indicates non-significant; p < 0.01, ∗∗; p < 0.001, ∗∗∗).

(A) VA10 cells were stretched for 6 and 24 h with (+) or without (−) 100 nM 1,25D3, (B) 2 mM PBA and (C) co-treated with vitamin D3 and PBA as shown in the figure. The mRNA expression of CAMP was assessed with qRT-PCR (n = 3, mean ± S.E.). Relative expression levels (y-axis) in static cells were defined with an arbitrary value of ‘1’ and changes relative to this value in stretched/treated samples are represented. (D) Similarly, the BCi cells were stretched for 6 h and 24 h with (+)/without (−) 100 nM 1,25D3 and the mRNA expression of CAMP was analyzed with q-RT PCR (n = 3, mean ± S.E.). Relative expression levels (y-axis) in static cells were defined with an arbitrary value of ‘1’ and changes relative to this value in stretched/treated samples are represented. (E) VA10 cells were treated with 20 nM 1,25D3 and stretched for 6 h and 24 h. LL-37 protein expression (red) was analyzed with immunofluorescence confocal microscopy. The cells were counterstained with nuclear stain DAPI (blue). The data is a representative of three independent experiments showing similar results. Bar = 100 µm. (F) Protein expression of cellular pro-LL-37 from stretched cells was also analyzed by Western blot analysis. VA10 cells were treated with 2 mM PBA, 20 nM 1,25D3 or co-treated with PBA and 1,25D3, followed by stretching for 24 h. GAPDH was used as a loading control. The Western blot is a representative of three independent experiments showing similar results (ns indicates non-significant; p < 0.05, ∗; p < 0.01, ∗∗; p < 0.001, ∗∗∗; p < 0.0001=∗∗∗∗).

Next, we screened for stretch mediated changes in toll-like receptor (TLR) expression in VA10 cells (Figs. 4A–4H). TLRs play an important role in activation of pro-inflammatory responses and have been shown to be modulated by mechanical stretching of cells (Takeda & Akira, 2005; Shyu et al., 2010). VA10 cells were stretched for 6 h and 24 h as described above. The mRNA expression of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR8 and TLR9 was analyzed with q-RT PCR. The mRNA expression of TLR3, TLR5 and TLR8 was reduced 6 h after stretch (Figs. 4C, 4E and 4G). Further, the mRNA expression of TLR2 (Fig. 4B) was increased and gene expression of TLR3 (Fig. 4C) and TLR9 (Fig. 4H) was decreased after 24 h stretch.

Our study has certain limitations. (1) The study was performed exclusively in cell lines. However, these respiratory cell lines (VA10 and BCi) have been shown to have primary cell like characteristics and have differentiation potential when cultured at an air–liquid interface (Halldorsson et al., 2007; Walters et al., 2013). They represent the upper airway lung epithelia. Primary human bronchial epithelial cells did not grow properly on collagen I coated bioflex silastic membranes and had to be excluded from this study. (2) The mechanism behind cyclic stretch mediated down-regulation of AMP expression needs to be elucidated and is a future area of interest. We hypothesize that stretch activated stress pathways (e.g., hypoxia related HIF-1α (Eckle et al., 2013; Fan et al., 2015)) could be involved in the observed down-regulation of AMP expression. Interestingly, acidification of cellular milieu upon cyclic stretch has been shown to promote bacterial growth in lung epithelial cells (Pugin et al., 2008). The relationship between stretch altered pH and its effects on AMP gene expression is also an area of interest.

In conclusion, our in vitro data shows that cyclic stretch down-regulates the expression of AMP cathelicidin in VA10 and BCi respiratory epithelial cells and activates a pro-inflammatory response in VA10 cells. These results could have clinical implications in regards to ventilator treatment of patients by identifying ways to increase the endurance of lung tissues to mechanical strain and preventing respiratory infections, encouraging further in vivo studies in this field.

Raw data

Acknowledgements

We would like to thank Jon Thor Bergthorsson and Katrin Birna Petursdottir for the help with analyzing flow cytometry data. We would like to specially thank Arí Jon Arason for introduction to the Flexcell tension system.

Gudmundur Hrafn Gudmundsson conceived and designed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper.

Data Availability

The following information was supplied regarding data availability:

The research in this article did not generate any raw data.

Funding

This project was supported by grants from LSH (Landspitali University Hospital), Össur hf and the Oddur Olafsson fund to Sigurbergur Karason. In addition this work was supported by funds from the University of Iceland and RANNIS. Nikhil Nitin Kulkarni was supported by University of Iceland grant for PhD students and RANNIS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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